The present invention relates to methods of screening compounds that modulate lipid kinases activity. The invention is more preferably based on the SPA technology to screen compounds that modulate the activity of lipid kinases, in particular membrane lipid kinases, cytosolic lipid kinases, secreted lipid kinases and more specifically sphingosine kinases. The invention also includes compositions, products, kits for use in performing the above methods, as well as the compounds identified by said methods, and their uses.
Lipid kinases are enzymes that catalyse the phosphorylation of lipids within cells. These enzymes, and the resulting phosphorylated lipids and lipid derived biologically active organic molecules, play a role in many different physiological processes, including cell proliferation, migration, adhesion, differentiation, activity, etc. A particular group of lipid kinases comprises membrane lipid kinases, i.e., kinases that catalyse the phosphorylation of lipids contained in or associated with cell membranes. Examples of such enzymes include phosphinositide(s) kinases (such as PI3-kinases); diacylglycerol kinases; and sphingosine kinases.
Sphingosine kinases (SPHK) convert the substrate sphingosine to sphingosine-1-phosphate (S1P). S1P is involved in various physiological processes, both within cells and upon release in extracellular medium. In particular, reported physiological roles of S1P within cells include the release of calcium from stores, activation of cyclin-dependent kinases, key signalling intermediate in Fc receptor initiated cascades, fMLP induced enzyme release, TNF-xcex1 induced (in endothelial cells) adhesion molecule expression, and depression of excitability in ventricular myocytes. Furthermore, when released from the cells, S1P is involved through specific G protein coupled (couple EDG-receptors) for instance in the control of cell proliferation, chemotaxis (attraction and activation of macrophages), cytoskeletal changes (stress fiber formation and cell shape changes, and secretion), cell attachment (fibronectin matrix assembly) and assembly and phosphorylation of paxillin and p125-FAK. Moreover, PDGF induces high levels of sphingosine kinase activity and S1P generation in platelets. In fact, S1P is released from activated platelets in large amounts. This could indicate a potential important role of S1P in inflammation following injury. More particularly, S1P could play an important role in early atherogenesis and fibrosis. Furthermore, sphingosine kinases activity play a major role in regulating calcium signals in mast cells activated via the high affinity IgE receptor (Fcxcex5RI), as well as in macrophages triggered by the high affinity IgG receptor (Fcxcex3RI), and activation of these cells are very important in allergic and auto-immune diseases, such us but not limited to, asthma and rheumatoid arthritis.
Accordingly, lipids, in particular membrane, cytosolic or secreted lipids, more specifically sphingosine-1-phosphate represent an interesting target for the development of drugs or pharmacologically active compounds. In particular, compounds having the ability to modulate the levels of S1P in cells would represent high potential compounds for the treatment of all diseases wherein S1P is involved such as cardiovascular diseases including atherosclerosis, thrombosis and dyslipidemia, diabetes including type I and type II diabetes and particularly type I diabetes, stroke, auto-immune and inflammatory diseases such as multiple sclerosis, psoriasis, epidermodysplasia verruciformis and inflammatory arthritis, allergic diseases such as dermatitis, T helper-1 related diseases, chronic obstructive pulmonary disease, asthma, cancer and neurodegenerative disorders.
The availability of assays suitable to screen compounds having such property would thus be of major interest. In this respect, the sphingosine kinase (SPHK) activity is classically measured using 33P or 32P incorporation, followed by lipid extraction and thin layer chromatography (TLC) separation of 33P or 32P sphingosine-1-P (A. Melendez et al., 2000). However, although this test is very sensitive, it is not suitable for high throughput screening, and no such test or method using TLC has been reported in the art, allowing efficient identification of lipid kinase modulators in high throughput format.
The present invention discloses compositions and methods for the screening of compounds that modulate, inhibit or activate the activity of lipid kinases, with reliability and efficacy. The methods according to this invention are simple, reliable, sensitive, convenient and economical, and allow screening of compounds, on a high throughput basis. In particular, the invention can be used to screen, in parallel, large numbers of compounds, including combinatorial libraries of compounds, to identify drug candidates or targets. This type of invention thus allows, for the first time, to screen active compounds using lipid kinases as targets, in particular SPHK, for the selection, improvement and/or development of therapeutically active products.
An object of this invention resides more specifically in a method of selecting or identifying a compound that modulates the activity of a lipid kinase, comprising (i) mixing (or contacting) the lipid kinase and a labeled lipid substrate thereof in the presence of a candidate compound and a source of phosphate, (ii) exposing the reaction mixture of (i) to a support material, wherein the support material binds the phosphorylated lipid (that has been phosphorylated by the lipid kinase) and essentially does not bind the unphosphorylated lipid, and (iii) assessing the amount of phosphorylated lipid bound to the support.
The support material may be composed of or comprise various elements, such as polymers, gels, glass, artificial or organic elements, etc . . . and more precisely components selected from yttrium-silicate, yttrium-oxyde or polyvinyltoluene (PVT). The support may further comprise (poly)acrylamide, agarose, sepharose or polystyrene or may be further functionalized, and may be shaped into various forms, including beads.
According to preferred embodiments, the method uses the scintillation proximity technology (SPA). In said embodiments, the support material further comprises a scintillant, which can be excited upon binding of the phosphorylated, (radio)labeled lipid substrate.
In a typical embodiment of the invention the lipid substrate is in a micelle and the source of phosphate is ATP.
The lipid kinase is a cell or membrane extract or may be purified. The lipid kinase may also be is a recombinant enzyme.
In a preferred embodiment, the reaction mixture in (i) comprises:
0.01-10 xcexcM of unlabeled lipid
0.01-10 xcexcCi of radio-labelled lipid,
0.1 to 5% of detergent, such as Triton, or neutral lipid, such as phosphatidyl serine or cardiolipine, or serum proteins from mammalian origin such as BSA (bovine serum albumin), HAS (human serum albumin) or FBSA; or a mixture thereof,
0.1 xcexcM to 1 mM of phosphate source, such as ATP, and
the desired amount of total proteins of a cell preparation comprising a lipid kinase (or any composition or material comprising the same) at the appropriate dilution.
In this regard, a more particular aspect of this invention resides in a method of selecting or identifying a compound that modulates, inhibits or activates the activity of a lipid kinase, comprising (i) mixing the said lipid kinase and a labeled lipid substrate thereof in the presence of a candidate compound and a source of phosphate, (ii) exposing the reaction mixture of (i) to beads, wherein the beads bind the lipid in phosphorylated form and essentially do not bind the lipid in unphosphorylated form, the beads further comprising a scintillant which is excitable by the labeled lipid upon binding thereof to the beads, and (iii) assessing the activity of the compound by assessing the scintillation of the beads.
The invention can be used for selecting, identifying, characterizing, improving, comparing, etc . . . compounds that modulate, inhibits or activates the activity of lipid kinases. The invention is more particularly suited for screening modulators of membrane lipid kinases, cytosolic lipid kinases or secreted lipid kinases, even more preferably sphingosine kinases.
A further object of this invention resides in a kit for use in the above screening assay, comprising a labelled lipid and/or a support as defined above.
A further object of this invention resides in the use of compounds selected or identified using the above methods, for pharmaceutical, therapeutical or experimental purposes.